CN103834640B - A kind of Chloroplast gene sequence to micro- method and correlation intended and foreign DNA is imported in ball algae chloroplast - Google Patents
A kind of Chloroplast gene sequence to micro- method and correlation intended and foreign DNA is imported in ball algae chloroplast Download PDFInfo
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Abstract
The present invention relates to technical field of biological genetic engineering, specifically a kind of Chloroplast gene sequence to micro- method and correlation intended and foreign DNA is imported in ball algae chloroplast.Gene order includes SEQID NO:Sequence, SEQ ID NO shown in 1:Sequence and insertion shown in 2 exogenous gene between the two.Concrete grammar will be including SEQ ID NO by electroporation:Sequence, SEQ ID NO shown in 1:The chloroplast transformation vector of the exogenous gene between the two of the sequence and insertion shown in 2 imports micro- plan ball frustule.The invention provides according to seven plants of micro- conservative genes for intending conservative gene and micro- plan ball algae chloroplast and diatom or Chloroplasts from Brown Algae genome between ball algae Chloroplast gene, can be used for the general Chloroplast Inheritance transformation system of the general Chloroplast Inheritance transformation system in micro- plan ball algae of design construction and the unicellular microalgae of multiple kinds.
Description
Technical field
The present invention relates to technical field of biological genetic engineering, specifically a kind of to outside importing in micro- plan ball algae chloroplast
The Chloroplast gene sequence of the method and correlation of source DNA.
Technical background
The energy and shortage of resources have become the sustained economic development of restriction China, affect the Major Strategic of China's national security
Problem.Based on biocatalyzer cleaning, it is efficient, renewable the features such as, biological manufacture can greatly reduce industrial development to fossil
Resource Dependence, reduces energy consumption, material consumption, water consume and waste discharge, significantly improves economic benefit, enhance one's market competitiveness.With stone
Change route to compare, biological manufacture can average energy-conservation 30-50%, reduce impact of the mankind to environment up to 20-60%.Sunlight is ground
Most sufficient clean energy resource on ball.Photosynthesis are the biological approaches that can uniquely catch solar energy, can also fix carbon dioxide,
Therefore it is to realize using solar energy and significantly reduce discharging CO2It is cleaned the effective way of production of energy.
Microalgae is that the most potential luminous energy realized in realistic meaning integrates one of cell factory of biological manufacture, and its advantage exists
In：Firstth, high efficiency light conversion of biomass：Microalgae（Including protokaryon cyanophyceae and Eukaryotic Algae）, as green higher plant, have
Than more complete photosynthesizer.The theoretical photosynthetic efficiency of microalgae reaches as high as 10%.Comparatively speaking, terrestrial crop one
As be only capable of 0.1 ~ 0.7%（C3 crops）Or 1.5 ~ 2.5%（C4 crops）Solar energy be converted into biomass.It can be seen that, microalgae conduct
Luminous energy is integrated the advantage of biological processing cell factory and is much larger than terrestrial plant.Additionally, some microalgae possess outstanding environmental resistance
Ability, can grow in fresh water, sea water, sanitary wastewater, industrial wastewater；The powerful growth when flue gas is passed through, can be outdoor
Scale evaluation.Secondth, efficient CO2It is fixed：During microalgae is dispersed throughout the fresh water such as earth ocean, river and lake and ocean water body and
Various soil（Including heat and the uncultivated environment of cold）In, or even in the extreme environment such as unfrequented south poles.It is annual by
The carbon dioxide that microalgae photosynthesis are fixed accounts for more than the 40% of global carbon dioxide fixed amount, follows in energy conversion and carbon
Play an important role in ring.Extensive microdisk electrode is expected to realize that industrialized mode is fixed solar energy and significantly reduces discharging CO2,
The clean energy resource of low-carbon (LC) is provided.3rd, efficient accumulation high-energy-density, high added value product：Microalgae metabolic pathway is enriched, with micro-
As a example by intending ball algae, it not only can accumulate oils and fatss under static state in a large number（Triglyceride, i.e. biodiesel）, can also accumulate
Microalgae albumen（Phycocyanin, forage protein, essential amino acids）, long-chain polyunsaturated fatty acid（AA、EPA、DHA）, plurality of color
Element（Astaxanthin, carotene, phylloxanthin）Contour value-added product, can be used for developing pigment, fertilizer, soil conditioner, antioxygen
Agent, food additive, cosmetics.
Ocean is micro- to intend ball algae（Nannochloropsis sp.), it is a kind of marine unicellular microalgae, belongs on taxonomy
In Phaeophyta (Phaeophyta), big eye algae guiding principle (Eustigmatophyceae), Dan Zhu algaes section（Monodopsidaceae）.It is micro-
Intend 2-5 μm of ball algae diameter, inorganization differentiation, ecologicaI distribution are extensive；Algae strain resource is being hidden with abundant；Evolutionary degree is unique,
It is an independent category in unicellular microalgae.There is research to compare Biomass, lipid content and the yield of 31 kinds of microalgae, wherein sea
The lipid production of foreign microalgae Nannochloropsis makes number one（Table 1）.Have found many strain Nannochloropsis all
With 1）Growth is rapid；2）A large amount of triglycerides are accumulated under the conditions of high light intensity or nutritional deficiency（TAG）The advantages of.Additionally,
It is large-scale at some（Such as Solix companies of the U.S.）And semi-industrial scale（Arizona State University, Chinese Academy of Sciences's Qingdao bioenergy
Source and process study institute）Find in outside scenery, when flue gas is passed through, Nannochloropsis（Such as
Nannochloropsissalina and Nannochloropsis oculata OZ-1）The powerful growth of energy, and can be static in growth
Phase accumulates substantial amounts of oils and fatss, possesses the potentiality of heavy industrialization culture.Additionally, micro- plan ball algae frond is small, rich in carbon aquation
Compound, protein and polyunsaturated fatty acid, are widely used in aquaculture as excellent bait；Micro- plan ball algae also contains
Diversified economy is worth higher pigment, such as zeaxanthin, Mylabris flavin and astaxanthin etc..It can be seen that, micro- plan ball algae is used as function base
Because group research and commercial applications have obvious advantage.
1 representative algae kind biomass accumulation of table, fat content and grease yield
Although micro- intend ball algae for the with the obvious advantage of luminous energy integration biological processing, its economic feasibility is still faced with huge
Challenge, main technical bottleneck include that micro algae growth density is low, and oils and fatss etc. are accumulated only under environmental stress conditions.Which is normal
Under physiological statuss, long-chain is accumulated（C16 and C18）With pole long-chain（C20, C22, C24 and C26）Fatty acid, Short-Chain Fatty Acids contain
Amount is low, causes the biodiesel cloud point of its production high, oxidizable, and the accumulation of long-chain fatty acid is synthetically produced to microalgae neutral fat
Suppress, these factors all become the micro- plan ball algae of restriction and are used for the critical bottleneck that luminous energy integrates biological production.
On the other hand, with the development of Microalgae biotechnology, micro- plan ball algae is used to build efficient microalgae reactor（Production is high
Value albumen, oils and fatss, pigment, antibacterial peptide）, exploitation oral type fisheries drug etc. aspect is with unique advantage.However, micro- plan ball algae gene work
The disappearance of journey, seriously constrains application of the ball algae as bioreactor in practice of micro- plan.
Can carry out in photosynthetic eukaryotic cell, nucleus, three kinds of organelles of chloroplast and mitochondrion respectively contain one
Set DNA molecular, constitutes genetic system that is not only independent but also connecting each other.From 20 century 70 technique for gene engineerings be born with
Come, the transformation technology for importing exogenous gene to nucleus has been widely used.However as the deep development of research, people
Gradually recognize that the genetic engineering operation in Matrix attachment region has the difficulty for being difficult to as follows overcome：1. nuclear genome is huge,
Background is complicated；2. the exogenous gene radom insertion for importing is in Matrix attachment region；3. exogenous gene expression efficiency is low, and expresses unstable
It is fixed；4. the insertion of exogenous gene can cause the variation of other character sometimes, so as to affect its economic worth；5. safety is bad,
Exogenous gene easily diffusion etc..These problems seriously govern improving for gene transformation technology and its answering in practice
With.
Chloroplast transformation technology can overcome disadvantage mentioned above.1988, Boynton etc. constructed the chloroplast of Chlamydomonas reinhardtii
Stable conversion system, demonstrates the feasibility of chloroplast transformation first（Boynton etc., Science, 1988）.After this, leaf
Green body transformation technology is successfully applied to the higher plants such as Nicotiana tabacum L.（Svab etc., Proc Natl Acad Sci, 1990）.With core
Conversion is compared, and chloroplast transformation technology has following features：1. exogenous gene can orient insertion；2. exogenous gene expression efficiency
High, variation is little；3. Chloroplast gene belongs to prokaryotic expression system, and multiple genes form polycistron, it is possible to achieve while table
Reach；4. biological safety is high.
Chloroplast transformation system includes following two key factors：One is suitable chloroplast transformation vector；Two is effective
Vectors into cells method.Suitable chloroplast transformation vector includes homologous fragment, selectable marker gene and possesses chloroplast
Startup, the regulating and controlling sequence of expiry feature.Principle of the chloroplast transformation based on homologous recombination, in chloroplast expression vector, external source base
Because both sides respectively have the homologous fragment of one section of chloroplast, length about 1Kb, longer homologous fragment are conducive to sending out for homologous recombination
It is raw.Meanwhile, in order to prevent exogenous gene from inserting the loss of the lethal effect and important sequence for causing, should also select suitable insertion
Site.Additionally, the copy number of Chloroplast gene is very high, need to set up efficient selection system, such as antibiotic and herbicide etc.
Screening, makes chloroplast transgenic cell realize homogeneity, thus selectable marker gene is the key for affecting chloroplast transformation efficiency
Factor.Exogenous gene is required to the promoter worked in chloroplast and terminator, ensure after exogenous origin gene integrator
Stability and high efficiency expression in chloroplast.
The conventional effective method by vector introduction Chloroplast gene is particle bombardment（Gene gun）, the method head
First in chlamydomonas（Chlamydomonas reinhardtii）In be successfully realized the conversion of Chloroplast gene, it is extensive afterwards
It is applied in the Chloroplast Inheritance transformation of higher plant and algae.Its ultimate principle is will be with target gene by dynamical system
The metallic particles of DNA（Goldc grains or tungsten particle）, cell is injected with certain speed, due to little particle penetration power by force, therefore is not required to remove
Cell wall and cell membrane and enter genome, so as to realize stable conversion.The transformation frequency of particle gun is big with acceptor specy, micro- bullet
Culture after little, bombarding pressure, the distance of prevention disk and gold grain, receptor pretreatment, receptor bombard has direct relation.Micro- bullet consumptive material
Tungsten powder or bronze are adopted often, both compare, and the injury that bronze may be than tungsten powder to cell is less with toxicity, and granular size
Unanimously.Tungsten powder is relatively cheap, but may toxic effect to some cells.Therefore, bronze is often used in chloroplast transformation,
A diameter of 4 μm of conventional bronze, less diameter are difficult to prepare, and minor diameter bronze can be because of space resistance, in the process of bombardment
It is middle that drift occurs.However, the bronze of this kind of size has the barrier that cannot go beyond in the application that microalgae Chloroplast Inheritance is transformed
Hinder.Most unicellular microalgae, diameter are less, and excessive bronze bullet causes the wound of unrepairable to cell, it is impossible to effectively
Screening obtains transformant.As micro- plan ball algae is a diameter of 2 ~ 5 μm, chloroplast diameter is only 1 μm or so, using the basic nothing of particle bombardment
Method is realized to the genetic engineering modified of its chloroplast.Electroporation（Electroporation）It is that exogenous gene is passed through into electric field
Effect, imports destination organization.Its principle is the moment of DC electric field effect, and surface of cell membrane produces hydrophobic or hydrophilic small logical
Road, this passage can maintain several milliseconds to several seconds, then voluntarily recover.Biomacromolecule such as DNA can pass through this during this period
Small passage enters cell.The method principle is simple, can effectively import exogenous gene, and efficiency is higher, with respect to other physics
And chemical conversion process, it is a kind of valuable and effective alternative method.In unicellular microalgae genetic engineering, especially leaf is green
In body conversion, electroporation is the only effective approach.However, the method is not yet used for unicellular microalgae Chloroplast Inheritance changing
In making.
Chloroplast genetic transformation technology is set up in micro- plan ball algae strong instrument can be provided for its genetic modification, can
The genetic resourcess bottleneck of the new industries such as biomass energy and material can fundamentally be broken through.Engineering algae strain is expected to building efficiently
Microalgae reactor（Production high level albumen, oils and fatss, pigment）, exploitation oral type fisheries drug etc. aspect performance unique advantage.Additionally, micro- plan
The foundation of ball algae chloroplast genetic transformation system can promote for its functional genomics in terms of research.The stable table of chloroplast
Key up to technology is to realize integration of the exogenous gene in Chloroplast gene and stable expression.Have one in micro- plan ball frustule
Individual cup type chloroplast, this structure are also beneficial to the foundation of chloroplast transformation system.But there is presently no with regard to micro- plan ball algae
Chloroplast gene sequence and the report of genetic transformation is carried out with micro- ball algae chloroplast of intending as object.
The content of the invention
It is an object of the invention to provide a kind of leaf to micro- method and correlation intended and foreign DNA is imported in ball algae chloroplast is green
Body genome sequence.For achieving the above object the present invention adopt technical scheme for：
A kind of sequence that foreign DNA is imported in micro- plan ball algae chloroplast, gene order include SEQ IDNO:Shown in 1
Sequence, SEQ ID NO:Sequence and insertion shown in 2 exogenous gene between the two.
The gene order includes SEQ ID NO:Micro- equivalent sequence intended in ball algae Chloroplast gene, SEQ shown in 1
ID NO:Micro- equivalent sequence intended in ball algae Chloroplast gene and insertion exogenous gene between the two shown in 2.It is described outer
Source gene is one or more genes.
The gene order has the DNA fragmentation of the applicable promoter, terminator and selected marker of chloroplast expression.
Using intending the method that the gene order of foreign DNA imports foreign DNA is imported in ball algae chloroplast to micro-, worn by electricity
Hole method will be including SEQ ID NO:Sequence, SEQ ID NO shown in 1:Sequence and insertion shown in 2 external source base between the two
The chloroplast transformation vector of cause imports micro- plan ball frustule.
Will be including SEQ ID NO by electroporation:Micro- equivalent sequence intended in ball algae Chloroplast gene, SEQ shown in 1
ID NO:The chloroplast of micro- equivalent sequence intended in ball algae Chloroplast gene and insertion exogenous gene between the two shown in 2
Conversion carrier imports micro- plan ball frustule.
The inventive method successfully constructs micro- chloroplast expression system for intending ball algae.Can effectively will be outer by the present invention
Source gene recombinaton is in micro- Chloroplast gene for intending ball algae, and obtains transgenic mutation algae strain by screening.
Compared with prior art, present invention achieves micro- key breakthrough for intending ball algae technique for gene engineering, with having as follows
Beneficial effect：
1. the present invention builds leaf green using two sections from micro- homologous recombination fragment for intending ball algae Chloroplast gene sequence
Exogenous gene can be imported micro- plan ball algae chloroplast by body site-directed transformation carrier, conversion carrier, and be allowed to green in micro- plan ball algae leaf
The expression of efficient stable in body.
2. the present invention is provided to micro- Chloroplast gene collection of illustrative plates and sequence for intending the transformation of ball algae chloroplast.Using chloroplast
Genome sequence can explain some closely related with photosynthesis albumen, enzyme and associated regulatory mechanism, and for improveing life
Thing（Such as plant, microorganism（Such as antibacterial, archeobacteria, cyanophyceae, single celled eukaryotic microalgae etc.））Economical character（Improve light energy absorption
With transformation efficiency etc.）.
3. the invention provides many plants of micro- conserved sequences and micro- plan ball algae and silicon intended between ball algae Chloroplast gene
Algae or the conserved sequence of Chloroplasts from Brown Algae genome, can be used to building the general Chloroplast Inheritance transformation system of micro- Sphaerellopsis or
Suitable for the general Chloroplast Inheritance transformation system of the unicellular microalgae of multiple kinds.
4. the present invention and can realize external source base by fixed point integration of foreign gene to micro- Chloroplast gene for intending ball algae
The stable expression of cause, using unicellular micro- physiological property intended under the induction of ball algae fluorescence, is used using green fluorescent protein GFP genes
In the structure of chloroplast transformation system.
5. the present invention intends sensitivity of the ball algae to bleomycin using micro-, using a kind of feasible selectable marker gene-choosing
Select force combination（Ble genes-bleomycin）For the structure of chloroplast transformation system.
6. the present invention can make exogenous gene it is micro- plan ball algae chloroplast in high efficient expression, and can accumulate in a large number destination protein,
Biological preparation（Auxin, interferon, serum albumin, insulin etc.）And vaccine（Cholera, rabies, anthrax, tetanus, Ah
Meter Ba etc.）.
7. the transgenic engineering microalgae for being built using the present invention, can possess more superior scale evaluation character, such as pest-resistant
Property, disease resistance, the drought-enduring, antiweed of salt resistance etc..Can be used to design and build the inexpensive specular removal for specific engineering microalgae
Reaction facility, reduce scale evaluation cost.
8. using the present invention, transplantation and build production terpenoid, long chain hydrocarbons, the biological elements of long-chain fat alcoholic compound and
Module, sets up cell factory for producing the high-energy-density hydrocarbon fuel such as long chain hydrocarbons, long-chain fatty alcohol, pigment, protein.Obtain
Obtain light-use to improve with carbon sequestration efficiency and the optimization of product approach and controllable new photosynthetic production cell factory.
9. micro- plan ball algae accumulates high added value substrate（Astaxanthin, plant sterol, docosahexenoic acid）, by the present invention
The transgenic engineering microalgae of structure, can further improve the content of high added value substrate.
10. micro- plan ball algae has the characteristics of photosynthetic efficiency is high, breeding is fast, environmental suitability is strong, builds by the present invention
Transgenic engineering microalgae, can further improve its photosynthetic efficiency, such that it is able to effective fixed carbon dioxide, reduce in air
The concentration of carbon dioxide.
11 micro- ball algae genomic sequences of intending have been determined, and can carry out functional genome to micro- plan ball algae by the present invention
Learn research.
Description of the drawings
Fig. 1 determines figure for micro- plan ball algae total protein concentration provided in an embodiment of the present invention.
Fig. 2 is micro- plan ball algae restriction modification system qualification figure provided in an embodiment of the present invention（Wherein, the total egg of micro- plan ball algae
In vain to plasmid p124S and pGenD without any enzyme action effect, it is seen then that there is no Restriction Enzyme cutting system in micro- plan ball algae.）.
Fig. 3 is micro- plan fatality rate figure of the ball algae under different voltages provided in an embodiment of the present invention（Wherein, respectively with 0v/
Cm, 600v/cm, 900v/cm, 1100v/cm, 1300v/cm, 1600v/cm, are shocked by electricity, and wherein electric capacity is set to 50 μ F, often
Individual process 3 is parallel.）.
Fig. 4 is micro- plan ball algae OZ-1 Chloroplast gene physical maps provided in an embodiment of the present invention.
Fig. 5 be many plants provided in an embodiment of the present invention it is micro- intend ball algae Chloroplast gene conservative genes and micro- plan ball algae and
Diatom or the conservative gene of Chloroplasts from Brown Algae genome.
Fig. 6 plasmid pXJ01 physical maps provided in an embodiment of the present invention.Wherein, Homologous integration site is light dependent/non-dependent
Protochlorophylid reductase（light-indepen-dent protochlorophyllidereductase）ChlL bases
Cause；Promoter is ribulose-1,5-bisphosphate, 5- diphosphonic acid carboxylases/oxygenase（Ribulosebisphosphate carboxylase
oxygenase）5 '-UTR of large subunit rbcL；3 '-UTRs of the terminator for chloroplast psbA.
Fig. 7 is that transgenic provided in an embodiment of the present invention is micro- intends ball algae shows fluorescent microscopy images（Wherein,（a）~（d）For 458nm
Picture under exciting light；（e）Picture under visible ray.）.
Fig. 8 is that transgenic provided in an embodiment of the present invention is micro- intends ball algae laser co-focusing figure（Wherein,（a）Collection red light；
（b）Collection green light；（c）Red and green light is gathered simultaneously.）.
Specific embodiment
The present invention obtain micro- plan ball algae chloroplast transformation algae strain, refer to using electric shocking method or other be introduced directly into method,
Intend the vector introduction of the micro- plan ball algae chloroplast homologous recombination fragment containing the present invention micro- in ball algae chloroplast, by homologous heavy
Group, the fixed point of the DNA fragmentation with exogenous gene is inserted in micro- plan ball algae Chloroplast gene.
The method for importing foreign DNA：
（1）Using SEQ ID NO:The equivalent sequence of the Chloroplast gene of sequence or its about 1Kb shown in 1, SEQ ID
NO:The equivalent sequence of the Chloroplast gene of sequence or its about 1Kb and exogenous gene constructed dna recombinant fragment shown in 2；（2）Will
DNA recombinant fragments are inserted in a kind of cloning vehicle；（3）Recombiant plasmid is imported into micro- plan ball frustule；（4）Homogeneity screening is obtained
Transgenic is micro- to intend ball algae.
The recombinant dna fragment with exogenous gene has promoter, terminator and the selected marker that chloroplast is suitable for；
The described SEQ ID NO for building chloroplast vector:1,2 equivalent sequence includes green according to micro- plan ball algae leaf
The intergenic region suitable for chloroplast homologous recombination, pseudogene area, function Yong Yu area, repetition designed by body genome sequence
Fragment area etc. is all recombinated after neither cause the important sequence of original Chloroplast gene to lose, will not disturb again and just close on gene
The section of Chang Gongneng.
The described SEQ ID NO for building chloroplast vector:1,2 equivalent sequence includes green according to micro- plan ball algae leaf
Functional gene area suitable for chloroplast homologous recombination designed by body genome sequence etc. can be used for functional genomicses research
Site.
The described cloning vehicle for building chloroplast vector is known any cloning vehicle, such as pUC18,
PUC19, pUC118, pUC119, pBluescript SK etc.）.
The described promoter for building chloroplast vector includes that micro- rbcL for intending ball algae Chloroplast gene source is opened
Mover and all DNA sequence for possessing chloroplast startup function.
The DNA sequence for possessing chloroplast startup function includes all of the sources such as plant, microalgae, virus, prokaryotic micro-organisms
Possesses the DNA sequence of chloroplast startup function.
Described terminator includes micro- psbA terminators for intending ball algae Chloroplast gene source and all to possess leaf green
The DNA sequence of body expiry feature.
The described DNA sequence for possessing chloroplast expiry feature include it is micro- plan ball algae Matrix attachment region, Chloroplast gene with
And all DNA sequence for possessing expiry feature in the source such as plant, microalgae, virus, prokaryotic micro-organisms.
The DNA fragmentation of described selected marker includes green fluorescent protein GFP genes and all with indicative function
Reporter gene.
The DNA fragmentation of described selected marker includes blasticidin resistance gene ble genes and all corresponds to micro- plan
The sensitive antibiotic of ball algae, the gene of pesticide.
The DNA fragmentation of described selected marker includes replying micro- gene code sequence for intending ball algae mutant original traits
Row.
The described exogenous gene for chloroplast transformation is arbitrary genes of interest, can be a gene or multiple
Gene；The gene of high level albumen, animal vaccine is encoded such as；Encoding regulator is micro- to intend ball algae growth, metabolism, photosynthesis or high additional
Value substrate（Neutral fat, astaxanthin, docosahexenoic acid）The regulatory factor of accumulation, albumen or enzyme.
The effect reached by the method for the present invention and the method is described in further detail with reference to case study on implementation.
Embodiment：Realize green fluorescent protein GFP genes in micro- stable expression intended in ball algae chloroplast
First, micro- checking for intending ball algae restriction modification system
There is narrow spectrum restriction endonuclease for phage-resistant invasion in bacterial cell, the system by into
For restriction modification system.The system is there is also in cyanophyceae.Single celled eukaryotic microalgae evolutionary degree is unique, if there is also similar
Restriction modification system.For this purpose, being verified using following steps：
（1）Micro- plan ball algae total protein extraction：The micro- plan ball algae of culture is to logarithmic (log) phase, 5000g, 10min, 4 DEG C of collection fronds；
5ml PENA solution（10mM K3PO4,10mM EDTA,50mM NaCland a teblet of aprotinin per
50ml,pH 7.0）Rinsing 2 times；Frond is resuspended in into 1ml PENA solution；100mg beades are added, be acutely vortexed 1min；
4000g, 15min, 4 DEG C of centrifugation;Supernatant is taken, equal-volume glycerol and 1/5 volume 1mg/ml bovine serum albumin, -20 DEG C of preservations are added
It is standby.
（2）Micro- plan ball algae total protein concentration is determined：Draw standard curve；10 times dilute micro- plan ball algae total protein liquid storage；Survey
Determine diluent OD260；Protein concentration is calculated for 30mg/L（Fig. 1）.
（3）It is prepared by carrier DNA：37 DEG C of digestion pGenD4h of 37 DEG C of digestion p124S 4h of BamHI, XbaI；Produced using PCR
Thing QIAquick Gel Extraction Kit（Qiagen companies）Reclaim fragment.
（4）Micro- plan ball algae total protein digestion process linearisation p124S and pGenD carrier：
Enzyme action system is as follows：It is micro- to intend ball algae total protein suspension X μ l, 1 μ l of Fermentas buffer (fast digest),
Linearized vector（6μg/μl）0.5 μ l, use dd H2O polishings are to 10 μ l.
37 DEG C of Jing is overnight processed, agarose gel electrophoresiies detection digestion effect.As shown in Fig. 2 micro- plan ball algae total protein pair
Plasmid p124S and pGenD is without any enzyme action effect, it is seen then that there is no Restriction Enzyme cutting system in micro- plan ball algae, without the need for being used for
The carrier of conversion carries out modification protection.The corresponding enzyme action system such as following table of each swimming lane in Fig. 2.
| Plasmid | 5μl | 3μl | 1μl | 1 μ l (10 times of diluents) | 1 μ l (100 times of diluents) |
| p124S | ① | ③ | ⑤ | ⑦ | ⑨ |
| p124S | ② | ④ | ⑥ | ⑧ | ⑩ |
| pGenD | a | c | e | g | i |
| pGenD | b | d | f | h | j |
2nd, micro- plan ball algae Chloroplast gene is determined and is spliced
Using second filial generation sequencing technologies Illumina determine 7 kinds it is not of the same race, of the same race do not belong to together it is micro- plan ball algae
N.oceanica OZ-1, N.oceanica CCMP531, N.granulata CCMP529, N.oculataCCMP525,
N.limnetica CCMP505, N.gaditana CCMP527, and the Chloroplast gene sequence of N.salinaCCMP537（Table
2）, large-scale checking has been carried out to spliced sequence by PCR and traditional Sanger sequencing, it was confirmed that splicing sequence
Reliability and accuracy, and polishing breach, are annotated using Dogma after splicing, and carry out manual synchronizing.
The different micro- plan ball algae Chloroplast gene sequence informations of table 2
| Kind | Size (bp) | G+C contents (%) | Number gene | TRNAs numbers |
| N.oceanica OZ-1 | 117548 | 33.6 | 122 | 28 |
| N.oceanica CCMP531 | 114614 | 33.0 | 121 | 28 |
| N.granulata CCMP529 | 116056 | 33.2 | 122 | 28 |
| N.oculata CCMP525 | 110774 | 32.7 | 115 | 26 |
| N.limnetica CCMP505 | 89751 | 33.0 | 92 | 20 |
| N.gaditana CCMP527 | 117548 | 33.6 | 122 | 28 |
| N.salina CCMP537 | 107159 | 33.1 | 122 | 28 |
3rd, micro- amplification for intending ball two sections of chloroplast homologous recombination fragments of algae and clone
Design and synthesize following two pairs of primers, wherein primer SEQ ID NO:3 and SEQ ID NO:4 amplified production is
SEQ ID NO:1；Primer SEQ ID NO:5 and SEQ ID NO:6 amplified production is SEQ ID NO:2.
With micro- plan ball algae genome DNA as template, Jing primer SEQ ID NO:3 and SEQ IDNO:4 enter performing PCR amplification,
Response procedures are：94 °C of 5min denaturations；94 °C of 30sec, 58 °C of 30sec, 72 °C of 1min, totally 30 circulations；72°C
7min extends.Pcr amplification product is about 952bp, as fragment SEQ ID NO:1, purified pcr product is connected to pMD-18T loads
Body, converts escherichia coli, extracts recombiant plasmid, with I enzyme action of Kpn I and Xho, the pBluescript with the same enzyme action of process
SK carriers connect, and obtain containing fragment SEQ ID NO:1 recombiant plasmid pSK-1.
With micro- plan ball algae genome DNA as template, Jing primer SEQ ID NO:5 and SEQ IDNO:6 enter performing PCR amplification,
Response procedures are：94 °C of 5min denaturations；94 °C of 30sec, 58 °C of 30sec, 72 °C of 1min, totally 30 circulations；72°C
7min extends.Pcr amplification product is about 1038bp.As fragment SEQ ID NO:2, purified pcr product is connected to pMD-18T
Carrier, converts escherichia coli, extracts recombiant plasmid, with I enzyme action of BamHI and Sac, is connected with the pSK-1 of the same enzyme action of process,
Obtain containing fragment SEQ ID NO:1 and SEQ ID NO:2 recombiant plasmid pSK-2.
SEQ ID NO:1
5’-GTAAACCAAGGTCGTTCTCCAGTTGGTAAACGTGATGCTTCAATTGGTGGAAACCCAAACCCAGCT TCTTTAAAGTTTCAATCAAATACCTAGAATTTATTTCTAGATAGTATTAAAATACATCTTAATTCATATCCTTTTAC TTTTACTTGAAGGGTATGATCTTAGGAGAGATGGCAGAGTGGTCGATTGCGTCTGACTTGAAATCAGAAGAACTAGG AATGGTTCCGTGGGTTCGAATCCCACTCTCTCTTCTCAATATTTTTATTAAAAATATAGGTTGATTCTTTTGATTTA ACTTTTTTCTATTAAAATATCTAATAGAACATTTCAAAAGTATTGTTTGGTTAACCTAAACCAGTTTATAATCTTTT AACTAAAGAGGTATATATGTTAGTACTAAAAATAGCAGTTTACACAGTTGTTAGCTTTTTCGTTTATCTATTTTGGT TTGGATTTATTTCAAATGACCCGTCACGTAACCCTACACAGAATATTAATAACTAATTAAAAGTTTGGTATTTATCA TAAGGCATATTAAATAAATAGAAATAATGCGCCTTATGATAAACGTAAATAAGTGCTATATATAAATAAAAGGGCAG TTAGCTCAGCGGTAGAGCTTCTGCCTTACAAGCAGAAGGCCACAGGTTCAAATCCTGTACTGCCCATAGGGCTCATC GTCTAAGGGATTAGGACAGAAACCTTCTAAGTTTCTAATGTAGGTTCGAATCCTACTGGGCCTAAGACGTACTGAGT ATAAAAAATTAAACATGATATTTTAAGGGTTACAGATAAACAAATGTTTTTGAGAAGATACTCTTTACTCCCAGAAT TTAAATACTAGTTGTCTGATTTTTTAACTCTGACACTCTAGACCTTATATTATAGTATTTTATGAGCAATTTATAAA AAATAAATTTGCGGTTACTTCTAGGAG
（a）Sequence signature：
● length：940bp
● type：Base sequence
● chain：It is single-stranded
● topological structure：Linearly
（b）Molecule type：Double-stranded DNA
（c）Assume：It is no
（d ）Antisense：It is no
（e）Initially originate：Micro- plan ball algae
（f）Specificity title：Protochlorophylid reductase (the light-indepen- of light dependent/non-dependent
Dentprotochlorophyllide reductase) 5 ' terminal sequences of chlL
SEQ ID NO:2
ATCCGAAGCATGATAGCACTTTTACATTAACAGGATTTTTAATTCCAACAATTATTGATACATTACAAGTCAAAGAT
TACCACTATGAGGATGTTTGGCCTGAAGATGTAATTTATAGAGGATTTAATGGTGTAGATTGTGTTGAGCTGGTGGA
CCGCCAGCTGGAGCTGGTTGTGGTGGTTACGTCGTTGGTGAAACTGTTAAACTTTTAAAAGAGCTTAATGCATTTGA
TGAATACGATGTTATTCTTTTTGATGTATTAGGTGATGTAGTTTGCGGTGGATTTGCAGCACCATTGAATTATGCGG
ATTACTGTTTAATTGTTACAGACAATGGCTTTGATGCCTTATTTGCCGCAAATAGAATTGCAGCTTCGGTTCGCGAA
AAAGCTAGGACTCACAGTTTAAGATTAGCTGGTTTAATAGGTAATCGAACTGCAACACGTGATTTAATTGTAAATAT
ATTCAAACTGTACCAATCCCAGTTCTTGAAGTTTTGCCACTAATTGAAGATATACGGGTTTCAAGAATTAAAGGTAA
AACGCTTTTTGAGATGTCTATAATTGATCCCTCATTGGGTACATTTGTGATTATTACTTAAATATTGCAGATCAACT
TATAGCTAACCAGAAGGTGTAATTCCAAAAGAATCAGCTGATCCGTGAATTATTTACTCTTTTATCTGATTTTTATT
TAAAACCCTCGGATACAGAACAAAATTTAGACAATATGGAGATGAATCTTTTTAATAATTAAATTACGTCTTAATGA
ATAAAAAAATTAAAAAGGAATTTAAATAATATGAATACAGAACAAGGTTCATTAATTAATTCTAATTCCATTACTTT
TGAATGCGAAACTGGTAATTATCATACATTTTGTCCTATAAGTTGTGTTGCTTGGTTATATCAAAAAATTGAAGACA
GTTTCTTTTTGGTAATTGGAACAAAAACGTGTGGCTATTTCTTACAGAATGCATTAGGGGTAATGATATTTGCTGAA
CCAAGGTACGCAATGGCTGAACTTGAAGAAGCTG-3’
（a）Sequence signature：
● length：1038bp
● type：Base sequence
● chain：It is single-stranded
● topological structure：Linearly
（b）Molecule type：Double-stranded DNA
（c）Assume：It is no
（d）Antisense：It is no
（e）Initially originate：Micro- plan ball algae
（f）Specificity title：Protochlorophylid reductase (the light-indepen- of light dependent/non-dependent
Dentprotochlorophyllide reductase) 3 ' terminal sequences of chlL
SEQ ID NO:3
ggtaccgtaaaccaaggtcgttctcca
SEQ ID NO:4
ctcgagctcctagaagtaaccgcaaat
SEQ ID NO:5
ggatccatccgaagcatgatagcactt
SEQ ID NO:6
gagctccagcttcttcaagttcagcca
4th, contain green fluorescence protein gene（gfp）Micro- plan ball algae chloroplast site-specific conversion carrier pXJ01 structure
The structure of carrier pXJ01 is based on above-mentioned recombiant plasmid pSK-2.Concrete building process is as follows.（1）Design
And synthetic primer.Wherein, primer SEQ ID NO:7 and SEQ ID NO:8 amplified production is 5 ' rbcL sequences, is from micro-
Intend the promoter with chloroplast startup function of ball algae chloroplast；Primer SEQ ID NO:9 and SEQ ID NO:10 amplification
Product is gfp genes, is green fluorescence protein gene；Primer SEQ ID NO:11 and SEQ ID NO:12 amplified production is
3 '-psbA, are from micro- terminator with chloroplast expiry feature for intending ball algae chloroplast.（2）HindIII and XhoI is double
5 ' rbcL of enzyme action pSK-2 and PCR primer, after purification digestion products, overnight connects, and converts escherichia coli, extracts recombiant plasmid, life
Entitled pSK-3.（3）With EcoRV and HindIII double digestions pSK-3 and PCR primer gfp, after purification digestion products, overnight connect,
Conversion escherichia coli, extract recombiant plasmid, are named as pSK-4.（4）With EcoRV and EcoRI double digestions pSK-4 and PCR primer
3 '-psbA, after purification digestion products, overnight connect, and convert escherichia coli, extract recombiant plasmid, as pXJ01（Referring to sequence
Table 3）.
SEQ ID NO:7
ctcgaggcttacttattagccaccacctaca
SEQ ID NO:8
aagcttttaggactccttttatatagcagtaaa
SEQ ID NO:9
aagcttatggtgagcaagggcgagga
SEQ ID NO:10
gatatcttacttgtacagctcgtccatgcc
SEQ ID NO:11
ggatccccgtatctaatgttcttccag
SEQ ID NO:12
gagctcaattggtcaccctgatggtat
5th, electroporation parameter optimization
Micro- plan ball algae OZ-1（Nannochloropsis OZ-1）It is inoculated in f/2 culture fluid, 25 °C of aerobic culture are to extremely
Logarithmic growth early stage（1-3×107cells/mL）, 6000g centrifugation 5min abandon supernatant, and 375mM Sorbitol is rinsed 2 times, uses Pyrusussuriensiss
Alcohol adjusts cell concentration to 2 × 108cells/mL.Concentration frond is packed as into the aliquot of 200 μ l, per part adds at 1 μ l degeneration
The salmon sperm dna of reason（15 μ g/mL, 95 °C are processed 1 minute), after mixing, ice puts 10 minutes.Mixture is transferred to into electric shock cup, point
Not with 0v/cm, 600v/cm, 900v/cm, 1100v/cm, 1300v/cm, 1600v/cm, shocked by electricity, wherein electric capacity is set to
50 μ F, each process 3 are parallel.Frond is transferred to into the fresh f/2 culture medium of 5mL immediately after electric shock.100rpm in 25 DEG C of shaking tables,
The low light level is recovered 48 hours.Coat f/2 flat boards.After 3 weeks, visible algae falls, and calculates fatality rate（Fig. 3）, wherein, semilethal voltage is about
For 1100v/cm.
6th, plasmid pXJ01 is imported micro- plan ball algae by electroporation
Take concentration and be about 1-3 × 107Micro- plan ball algae algae solution OZ-1 of the exponential phase of cells/mL
（Nannochloropsis OZ-1）, 6000g centrifugation 5min abandon supernatant, and 375mM Sorbitol is rinsed 2 times, thin with Sorbitol adjustment
Born of the same parents' concentration is to 2 × 108cells/mL.Concentration frond is packed as into the aliquot of 200 μ l, per part of 3 μ g linearized vector pXJ01 of addition
With the salmon sperm dna of 1 μ l degenerative treatments（15μg/mL）, after mixing, ice puts 10min.Mixture is transferred to into 2mm electric shock cups, with
2200V（HV）, 50 μ F are shocked by electricity, and frond is transferred to the fresh f/2 culture medium of 5mL immediately after electric shock.In 25 DEG C of shaking tables
100rpm, low light level recovery 48h.
7th, the observation of chloroplast transformation algae strain
Through micro- plan ball frustule of renewal cultivation, 10 are diluted to f/24Individual/mL, takes 10 μ l and sees under fluorescence microscope
Examine.Wherein, a length of 458nm of excitation light wave.Such as Fig. 7, the strain of wild type algae are presented redness because of its chlorophyll autofluorescence, transformant
Strong green fluorescence is sent because expression has green fluorescent protein.
Through micro- plan ball frustule of renewal cultivation, 10 are diluted to f/24Individual/mL, takes 10 μ l micro- in laser co-focusing
Microscopic observation.Wherein, a length of 458nm of excitation light wave.Such as Fig. 8 a, when only gathering red light, in the visual field, all microalgae cells are in
Existing redness, this is chlorophyll autofluorescence；When only gathering green light such as Fig. 8 b, in the visual field, only three cells are presented green, and this is
The fluorescence that green fluorescent protein sends；When gathering red and green light simultaneously such as Fig. 8 c, in the visual field, most cells are presented red, only
There are three cells that yellow is presented, red is leaf green autofluorescence, and yellow is the spontaneous red fluorescence of leaf green and green fluorescence egg
White green fluorescence is formed by stacking.This shows that pXJ01 has been incorporated in Chloroplast gene, and green in some positive algae strains
Color fluorescin successful expression.
In addition, according to the micro- characteristic for intending ball algae of difference, with reference to method and DNA provided by the present invention for converting, can
By the exogenous gene insertion SEQ ID NO of different qualities:Sequence shown in 1 and SEQID NO:Between sequence shown in 2, then
It is integrated in micro- plan ball algae, the present invention can be made to obtain higher utilization.
I.e. micro- to intend the choosing that ball algae is the ideal for carrying out biological diesel oil refining, the transgenic engineering built by the present invention is micro-
Algae, can further improve its oil and fat accumulation, promote the industrialization process of microalgae diesel oil；In addition, micro- plan ball algae is that accumulation is high attached
Value added substrate（Astaxanthin, docosahexenoic acid）, by the transgenic engineering microalgae that the present invention builds, further can improve
The content of high added value substrate；More go up micro- plan ball algae Matrix attachment region and Chloroplast gene sequence has been determined, by this
It is bright functional genomicses research to be carried out to which in micro- plan ball algae；Additionally, micro- plan ball algae non-coding RNA groups sequence is
Jing is determined, and can carry out functional genomicses research to which by the present invention in micro- plan ball algae；
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any spirit without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (2)
1. it is a kind of to micro- carrier for intending exogenous gene is imported in ball algae chloroplast, it is characterised in that the carrier includes SEQ
IDNO:DNA fragmentation, SEQ ID NO shown in 1:DNA fragmentation and insertion shown in 2 exogenous gene between the two；The load
Body also has micro- DNA fragmentation for intending promoter, terminator and selected marker that ball algae chloroplast expression is suitable for.
2. the method for the vector introduction exogenous gene described in a kind of utilization claim 1, it is characterised in that：Will by electroporation
Vector introduction described in claim 1 is micro- to intend ball frustule.
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